(1) Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of CD4+CD25+Foxp3+ regulatory T cells (Treg). Our recent studies have focused on defining the mechanism of action of the CD4+CD25+Foxp3+ Treg cells in vitro and on an analysis of their potential dysfunction in autoimmune disease: (1) We have analyzed the role of the transcription factor Helios (Ikzf2) in Treg function. Helios is a transcription factor belonging to the zinc finger containing Ikaros family comprised of five transcription factors-- Ikaros, Helios, Aiolos, Eos and Pegasus. Ikaros is expressed in all immune cells. The Helios gene (Ikzf2) encodes protein isoforms with zinc-finger domains exhibiting considerable homology to Ikaros family proteins. Human and mouse Helios share 97% identity in their amino acid sequences. In order to more thoroughly examine Helios expression and function, we generated a monoclonal antibody (22F6) to a large peptide within the non-conserved 110 amino acid region in Helios. When LN cells were gated on CD4+ cells and analyzed for Foxp3 and Helios expression, Helios was expressed on only 70% of Foxp3+ cells. As 22F6 was raised to a mouse peptide that differed from the human sequence by only two amino acids, we analyzed the reactivity of 22F6 in human cells. 22F6 also reacted with human PBMCs and was again, specific to the CD4+CD25hi Treg subset. Helios was expressed at the DN stage of thymic development. Specifically, Helios was expressed at low levels as early as DN1, but was expressed in 100% of DN2 and DN3 cells. By DN4, Helios expression had begun to decrease. In contrast, Foxp3 was not expressed at the DN stage and thus, Helios precedes Foxp3 expression. We also observed that the percentage of CD4+Foxp3+ Helios- cells was significantly lower in the thymus than in the periphery. Notably, there are no Foxp3+ Helios- cells at day 3 of life. Foxp3+ Helios- cells are absent in the neonate, do not significantly appear in the spleen until between day 7 and day 14 of life and do not reach the percentage observed in an adult until after the pups have been weaned. The observation that Foxp3+Helios- cells appear to arise in peripheral lymphoid tissue and do not acquire their full phenotypic profile until after weaning led us to examine the expression of Helios in TGF-beta induced iTreg cells. Both human and mouse iTreg were helios-. Collectively, these results demonstrate that Helios is a specific marker of thymic-derived Treg cells and raise the possibility that a significant percentage of Foxp3+ Treg cells are generated extra-thymically. (2) Stimulation of naive mouse CD4+Foxp3- T cells in the presence of TGF-beta results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced regulatory T cells (iTreg) is unstable, raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated ovalbumin-specific iTreg from mice expressing an anti-ovalbumin T cell receptor transgene (OT-II). Following transfer to normal C57BL/6 mice, OT-II iTregs maintained high levels of Foxp3 expression for 8 d. However, they rapidly lost Foxp3 expression upon stimulation when immunized with ovalbumin. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region within the Foxp3 locus. Administration of IL-2/antiIL-2 complexes expanded the numbers of transferred Foxp3+ iTreg in the absence of Ag challenge. Notably, when the iTreg were stimulated with antigen, treatment with IL-2/antiIL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the Treg-specific demethylated region. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-beta in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases. (3) Although the contributions of nTregs and iTregs to maintaining immune homeostasis remain to be determined, knowledge of how these cells are induced physiologically is key to understanding how T cell immunity is regulated as well as highly relevant for managing diverse human disorders. A previously unrecognized early event in T cell activation is the that both antigen-specific T cells and antigen presenting dendritic cells (DC) synthesize the alternative pathway complement components C3, C5, factor B, factor D, and the G-protein coupled receptors (GPCRs), C3a/C5a receptors (C3aR/C5aR). As a consequence of this local synthesis of complement, the anaphylotoxins, C3a and C5a, are produced and ligate C3aR and C5aR on the interacting on the T cell and DC surfaces in both a paracrine and autocrine fashion. In previous studies from the Medof lab, disabling C3aR/C5aR signaling resulted in a marked reduction in both the costimulatory and survival signals needed for effector T cell responses was observed. More importantly, C3aR/C5aR signaling was required for IL-6 dependent Th17 lineage commitment, as disabling C3aR/C5aR signaling markedly suppressed the IL-17 response and the induction of EAE. The requirement of C3aR/C5aR signaling for the production of IL-6 raised the possibility that inhibition of IL-6 production by blocking C3aR/C5aR signal transduction would lead to the production of iTreg as the induction of iTreg is inhibited in the presence of IL-6. In collaborative studies with the Medof lab, we demonstrate that blockade of C3aR/C5aR signaling during T cell activation results in the induction of iTreg in a TGF-beta-dependent fashion which manifest potent suppressor function both in vitro and in vivo.